Method for identifying olfactory receptor included in one olfactory cell

ABSTRACT

The present invention provides a novel method for identifying an olfactory receptor included in one olfactory cell. In the present invention, amplified is the cDNA derived from the mRNA of the one olfactory cell by a PCR method using a forward primer represented by SEQ ID: 01 and a reverse primer represented by SEQ ID: 02. Subsequently, determined is whether or not a gene sequence of the amplified cDNA is identical to one gene sequence included in gene sequences coding for olfactory receptors included in the mouse olfactory receptor group A. Finally, determined is that, if the gene sequence of the cDNA is identical to the one gene sequence in the previous step, the olfactory receptor included in the one olfactory cell is the olfactory receptor corresponding to the one gene sequence which is identical to the gene sequence of the cDNA in the previous step.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of PCT International ApplicationPCT/JP2012/004048 filed on Jun. 22, 2012, which claims priority toJapanese Patent Application No. 2012-064991 filed on Mar. 22, 2012. Thedisclosures of each of these applications including the specification,the drawings and the claims are hereby incorporated by reference intheir entirety.

INCORPORATION BY REFERENCE-SEQUENCE LISTING

The material contained in the ASCII text file named“P634278_(—)01_(—)566080_Sequence.txt” created on Feb. 22, 2013 andhaving a file size of 6167 bytes is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for identifying an olfactoryreceptor included in one olfactory cell.

2. Background of the Related Art

An olfactory receptor is a trimeric G protein-coupled receptor(hereinafter, referred to as “GPCR”). More particularly, the olfactoryreceptor is one kind of trimeric G protein-coupled seven-transmembranereceptors.

FIG. 4 shows a mechanism that a stimulus of an odor molecule to a cellmembrane is converted into an electric signal.

The olfactory receptor is a membrane protein which is expressed on thecell membrane. The cell membrane is mainly composed of a lipid bilayermembrane. The lipid bilayer membrane has a structure of two layers eachconsisting of phospholipid molecules lined with a high density. Thislipid bilayer membrane is shown in the center of FIG. 4 schematically.In FIG. 4, the outside of the cell is above the upper part of the lipidbilayer membrane. On the other hand, the inside of the cell is below thelower part of the lipid bilayer membrane. The trimeric G protein isplaced in the vicinity of the olfactory receptor.

The trimeric G protein is a heterotrimer is composed of an alpha subunit(Gαolf), a beta-subunit (Gβ), and a gamma subunit (Gγ). The cellcontains adenylate cyclase. In FIG. 4, the adenylate cyclase is referredto as “AC”. To be more exact, the adenylate cyclase is atransmembrane-type protein. A protein RTP1S has a function to assist theolfactory receptors to be expressed in the cell membrane. It is notedthat the protein RTP1S is not directly associated with the mechanism.

Next, the mechanism is described. The odor molecule binds to theolfactory receptor. The binding separates the trimeric G protein intothe alpha subunit (Gαolf) and a beta-gamma complex. The beta-gammacomplex consists of the subunit Gβ and the subunit Gγ. The separatedGαolf activates the adenylate cyclase (AC). The activated adenylatecyclase (AC) converts adenosine triphosphate (ATP) into cyclic adenosinemonophosphate (cAMP).

The cyclic adenosine monophosphate (cAMP) activates an ion channel, moreparticularly, for example, a cyclic nucleotide gated ion channel (CNG).The activation allows an ion to be transported from the inside of thecell to the outside of the cell, or from the outside of the cell to theinside of the cell. The degree of the transport of the ion can bemeasured as an electric signal.

The mouse contains approximately 1,300 kinds of the olfactory receptors.The olfactory receptor is referred to as “Olfr_n”. Generally, nrepresents a natural number. For example, an olfactory receptor having aname of “Olfr971” is known.

One olfactory cell includes one kind of the olfactory receptor. Oneolfactory cell does not include two or more kinds of the olfactoryreceptors.

When an olfactory receptor is not expressed in a cell transfected with agene sequence coding for an olfactory receptor, a skilled person who hasacquired one olfactory cell is not able to identify the olfactoryreceptor included in the one olfactory cell.

In order to solve this problem, Non Patent Literature 1 discloses amethod for identifying an olfactory receptor included in one olfactorycell among Olfr 480, Olfr 544, Olfr 545, Olfr 586, Olfr 642, Olfr 661,Olfr 672, Olfr 690, Olfr 744 and Olfr 749, using a pair of primersrepresented by SEQ ID: 03 and SEQ ID: 04, a pair of primers representedby SEQ ID: 03 and SEQ ID: 05, or a pair of primers represented by SEQID: 03 and SEQ ID: 06.

Non Patent Literature 2 discloses a method for determining whether ornot an olfactory receptor included in one olfactory cell is Olfr 16,using a pair of primers represented by SEQ ID: 07 and SEQ ID: 08.

Non Patent Literature 3 discloses a method for identifying an olfactoryreceptor included in one olfactory cell among Olfr 1056, Olfr 1366 andOlfr 1484, using a pair of primers represented by SEQ ID: 03 and SEQ ID:09.

CITATION LIST Non Patent Literatures

[Non Patent Literature 1]

Bettina Malnic, Junzo Hirono, Takaaki Sato, B. Buck, (1999) Cell, 96,713-723

[Non Patent Literature 2]

Touhara K., Sengoku S., Inaki K., Tsuboi A., Hirono J., Sato T., SakanoH., Haga T. (1999) Proc. Natl. Acad. Sci., 96, 4040

[Non Patent Literature 3]

Hamana H., Hirono J., Kizumi M., Sato T., Chem. Senses (2003) 28 (2):87-104.

BRIEF SUMMARY OF THE INVENTION Technical Problem

The purpose of the present invention is to provide a novel method foridentifying an olfactory receptor included in one olfactory cell.

Solution to Problem

The present invention is a method for identifying an olfactory receptorincluded in one olfactory cell among a mouse olfactory receptor group A;the method comprising steps of:

(a) obtaining the one olfactory cell;

(b) extracting mRNA contained in the one olfactory cell obtained in thestep (a);

(c) obtaining cDNA with reverse transcriptase from the mRNA extracted inthe step (b);

(d) amplifying the cDNA obtained in the step (c) by a PCR method using aforward primer represented by SEQ ID: 01 and a reverse primerrepresented by SEQ ID: 02; wherein

SEQ ID:01 is A (adenine) -T (thymine) -G (guanine) -K (guanine orthymine) -C (cytosine) -I (inosine) -T (thymine) -W (adenine or thymine)-I (inosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -M(adenine or cytosine) -G (guanine) -I (inosine) -T (thymine) -A(adenine) -Y (thymine or cytosine) -G (guanine) -T (thymine) -I(inosine) -G (guanine) -C (cytosine); and

SEQ ID: 02 is T (thymine) -C (cytosine) -Y (thymine or cytosine) -T(thymine) -K (guanine or thymine) -R (guanine or adenine) -T (thymine)-T (thymine) -Y (thymine or cytosine) -C (cytosine) -T (thymine) -I(inosine) -A (adenine) -G (guanine) -R (guanine or adenine) -C(cytosine) -T (thymine) -R (guanine or adenine) -T (thymine) -A(adenine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -A(adenine) -G (guanine) -I (inosine) -G (guanine) -G (guanine) -R(guanine or adenine) -T (thymine) -T (thymine)

(e) determining whether or not a gene sequence of the cDNA amplified inthe step (d) is identical to one gene sequence included in genesequences coding for olfactory receptors included in the mouse olfactoryreceptor group A;

(f) determining that, if the gene sequence of the cDNA is identical tothe one gene sequence in the step (e), the olfactory receptor includedin the one olfactory cell is the olfactory receptor corresponding to theone gene sequence which is identical to the gene sequence of the cDNA inthe step (e);

where the mouse olfactory receptor group A includes the following mouseolfactory receptors:

(Mouse Olfactory Receptor Group A)

Olfr1, Olfr8, Olfr20, Olfr22-ps1, Olfr29-ps1, Olfr30, Olfr33, Olfr44,Olfr53, Olfr76, Olfr155, Olfr156, Olfr159, Olfr209, Olfr290, Olfr291,Olfr295, Olfr314, Olfr327-ps1, Olfr329, Olfr332, Olfr361, Olfr368,Olfr373, Olfr382, Olfr437, Olfr457, Olfr472, Olfr476, Olfr480, Olfr482,Olfr484, Olfr498, Olfr502, Olfr505-ps1, Olfr508, Olfr538, Olfr544,Olfr694, Olfr695, Olfr697, Olfr706, Olfr713, Olfr715, Olfr721-ps1,Olfr735, Olfr802, Olfr877, Olfr914, Olfr921, Olfr945, Olfr951, Olfr971,Olfr988, Olfr992, Olfr994, Olfr995, Olfr1032, Olfr1082, Olfr1084,Olfr1095, Olfr1097, Olfr1120, Olfr1145, Olfr1152, Olfr1162, Olfr1170,Olfr1173, Olfr1328, Olfr1425, Olfr1502 and Olfr1504.

In one embodiment, a cell membrane of the one olfactory cell between thestep (a) and the step (b) is disrupted.

In one embodiment, the one olfactory cell is extracted from a samplesolution containing olfactory cells in the step (a).

In one embodiment, the mouse olfactory receptor group A is selected fromthe following mouse olfactory receptor group B:

(Mouse Olfactory Receptor Group B)

Olfr 1, Olfr 8, Olfr 20, Olfr 22-ps1, Olfr 29-ps1, Olfr 33, Olfr 44,Olfr 76, Olfr156, Olfr209, Olfr290, Olfr291, Olfr295, Olfr314,Olfr327-ps1, Olfr329, Olfr332, Olfr361, Olfr368, Olfr437, Olfr472,Olfr476, Olfr480, Olfr482, Olfr484, Olfr502, Olfr505-ps1, Olfr508,Olfr538, Olfr694, Olfr695, Olfr697, Olfr706, Olfr721-ps1, Olfr735,Olfr802, Olfr877, Olfr914, Olfr921, Olfr945, Olfr951, Olfr971, Olfr988,Olfr992, Olfr994, Olfr995, Olfr1032, Olfr1082, Olfr1084, Olfr1145,Olfr1152, Olfr1170, Olfr1173, Olfr1502 and Olfr1504.

Advantageous Effects of Invention

The present invention provides a novel method for identifying anolfactory receptor included in one olfactory cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic diagram of the example 1.

FIG. 2 shows an electrophoretic photograph according to the comparativeexample 1, the comparative example 2, the comparative example 3, and theexample 1.

FIG. 3 shows a schematic diagram of the reference example.

FIG. 4 shows a mechanism that a stimulus of an odor molecule to a cellmembrane is converted into an electric signal.

FIG. 5 shows SEQ ID: 03-SEQ ID: 06.

FIG. 6 shows SEQ ID: 07-SEQ ID: 09.

FIG. 7 shows SEQ ID: 10.

DETAILED DESCRIPTION OF THE INVENTION

The embodiment of the present invention is described below.

(Definition of Term)

The term “mouse olfactory receptor group A” used in the instantspecification means mouse olfactory receptors Olfr1, Olfr8, Olfr20,Olfr22-ps1, Olfr29-ps1, Olfr30, Olfr33, Olfr44, Olfr53, Olfr76, Olfr155,Olfr156, Olfr159, Olfr209, Olfr290, Olfr291, Olfr295, Olfr314,Olfr327-ps1, Olfr329, Olfr332, Olfr361, Olfr368, Olfr373, Olfr382,Olfr437, Olfr457, Olfr472, Olfr476, Olfr480, Olfr482, Olfr484, Olfr498,Olfr502, Olfr505-ps1, Olfr508, Olfr538, Olfr544, Olfr694, Olfr695,Olfr697, Olfr706, Olfr713, Olfr715, Olfr721-ps1, Olfr735, Olfr802,Olfr877, Olfr914, Olfr921, Olfr945, Olfr951, Olfr971, Olfr988, Olfr992,Olfr994, Olfr995, Olfr1032, Olfr1082, Olfr1084, Olfr1095, Olfr1097,Olfr1120, Olfr1145, Olfr1152, Olfr1162, Olfr1170, Olfr1173, Olfr1328,Olfr1425, Olfr1502 and Olfr1504.

The term “mouse olfactory receptor group B” used in the instantspecification means mouse olfactory receptors Olfr 1, Olfr 8, Olfr 20,Olfr 22-ps1, Olfr 29-ps1, Olfr 33, Olfr 44, Olfr 76, Olfr156, Olfr209,Olfr290, Olfr291, Olfr295, Olfr314, Olfr327-ps1, Olfr329, Olfr332,Olfr361, Olfr368, Olfr437, Olfr472, Olfr476, Olfr480, Olfr482, Olfr484,Olfr502, Olfr505-ps1, Olfr508, Olfr538, Olfr694, Olfr695, Olfr697,Olfr706, Olfr721-ps1, Olfr735, Olfr802, Olfr877, Olfr914, Olfr921,Olfr945, Olfr951, Olfr971, Olfr988, Olfr992, Olfr994, Olfr995, Olfr1032,Olfr1082, Olfr1084, Olfr1145, Olfr1152, Olfr1170, Olfr1173, Olfr1502 andOlfr1504.

In the instant specification, “Olfr” having “O” of a capital lettermeans an olfactory receptor.

In the instant specification, “olfr” having “o” of a small letter meansa gene sequence coding for an olfactory receptor.

A skilled person who has read the instant specification could identifyan olfactory receptor included in one olfactory cell among the mouseolfactory receptor group A. Unfortunately, a skilled person could NOTidentify an olfactory receptor, when the olfactory receptor included inone olfactory cell is not an olfactory receptor included in the mouseolfactory receptor group A (For example, when the olfactory receptor isOlfr 2).

Generally, an olfactory receptor is included in one olfactory cell insuch a manner that the olfactory receptor penetrates a cell membrane ofthe one olfactory cell.

(Step (a))

First, one olfactory cell is prepared. It is desirable that the oneolfactory cell is extracted from a sample solution containing olfactorycells. More particularly, the sample solution is supplied onto a petridish. One olfactory cell contained in the sample solution which has beensupplied onto the petri dish is found with a microscope. The oneolfactory cell thus found is collected into a capillary tube. In thisway, the one olfactory cell is isolated. The one olfactory cell isderived from a mouse.

(Step (b))

In the step (b), mRNA contained in the one olfactory cell obtained inthe step (a) is extracted. More particularly, the cell membrane of theone olfactory cell is disrupted with a cell lysate to extract the mRNAcontained in the one olfactory cell. The cell lysate is included in akit available from Takara bio Co., Ltd., as a commercial name: CellAmpWhole Transcriptome Amplification Kit Ver2.

(Step (c))

In the step (c), cDNA is obtained with reverse transcriptase from themRNA extracted in the step (b). To obtain the cDNA using the reversetranscriptase from the mRNA, used is a kit available from Takara bioCo., Ltd., as a commercial name: CellAmp Whole TranscriptomeAmplification Kit Ver2.

(Step (d))

In the step (d), the cDNA obtained in the step (c) is amplified by a PCRmethod using a forward primer represented by SEQ ID: 01 and a reverseprimer represented by SEQ ID: 02.

SEQ ID:01 is A (adenine) -T (thymine) -G (guanine) -K (guanine orthymine) -C (cytosine) -I (inosine) -T (thymine) -W (adenine or thymine)-I (inosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -M(adenine or cytosine) -G (guanine) -I (inosine) -T (thymine) -A(adenine) -Y (thymine or cytosine) -G (guanine) -T (thymine) -I(inosine) -G (guanine) -C (cytosine).

SEQ ID: 02 is T (thymine) -C (cytosine) -Y (thymine or cytosine) -T(thymine) -K (guanine or thymine) -R (guanine or adenine) -T (thymine)-T (thymine) -Y (thymine or cytosine) -C (cytosine) -T (thymine) -I(inosine) -A (adenine) -G (guanine) -R (guanine or adenine) -C(cytosine) -T (thymine) -R (guanine or adenine) -T (thymine) -A(adenine) -I (inosine) -A (adenine) -T (thymine) -I (inosine) -A(adenine) -G (guanine) -I (inosine) -G (guanine) -G (guanine) -R(guanine or adenine) -T (thymine) -T (thymine)

When the olfactory receptor included in the one olfactory cell is anolfactory receptor included in the mouse olfactory receptor group A, thecDNA is amplified in the step (d) by a PCR method. On the other hand,when the olfactory receptor included in the one olfactory cell is NOT anolfactory receptor included in the mouse olfactory receptor group A, thecDNA is NOT amplified in the step (d) by a PCR method. If the cDNA isamplified in the step (d), the next step (e) is carried out.

(Step (e))

In the step (e), it is determined whether or not the gene sequence ofthe cDNA amplified in the step (d) is identical to one gene sequenceincluded in gene sequences coding for the olfactory receptors includedin the mouse olfactory receptor group A.

More particularly, the gene sequence of the cDNA amplified in the step(d) is analyzed. Then, one gene sequence which is identical to theanalyzed gene sequence is found by a BLAST method from among a pluralityof known gene sequences olfrs, which are coding for olfactory receptorsOlfrs. Regarding the BLAST search method, refer to the following homepage: http://blast.ncbi.nlm.nih.gov/Blast.cgi. In addition, the genesequence coding for each of the olfactory receptors included in themouse olfactory receptor group A is known.

For example, when the gene sequence of the cDNA amplified in the step(d) corresponds with the gene sequence olfr 971 by the BLAST searchmethod, it is determined that the olfactory receptor included in the oneolfactory cell is Olfr 971.

(Step (f))

In this way, in the step (e), when the gene sequence of the cDNA isidentical to one gene sequence included in the gene sequences coding forthe olfactory receptors included in the mouse olfactory receptor groupA, it is determined that the olfactory receptor included in the oneolfactory cell is the olfactory receptor corresponding to the one genesequence which is identical to the gene sequence of the cDNA in the step(e).

EXAMPLES

The following examples describe the present invention in more detail.

(Preparation of the Sample Solution Containing Olfactory Cells)

One mouse C57/BL6J (female) was purchased from Japan SLC, Inc. The mousewas 3-5 weeks old.

A anesthetic agent (pentobarbital sodium, 100 microliters, availablefrom Kyoritsu Seiyaku Corporation) diluted tenfold with a saline(available from Otsuka Pharmaceutical Co., Ltd.) was injected into theabdominal cavity of the mouse using an injector having a needle of 1milliliter. The mouse was left at rest for five minutes.

After it was confirmed that the mouse was in the anesthetic state, themouse head was cut off with scissors.

In order to prevent the mouse tissue from being necrotized, the mousehead was immediately immersed in a sherbet of a Ca²⁺-free Ringer'ssolution prepared in a beaker having a volume of 50 milliliters. Themouse head was immersed in the sherbet for five minutes.

The Ca²⁺-free Ringer's solution had a composition shown in the followingTable 1.

TABLE 1 Chemical reagent Concentration NaCl 140 mM KCl 5 mM HEPES 10 mMEDTA 1 mM Glucose 10 mM Pyruvate sodium 1 mM

These chemical reagents contained in the Ca²⁺-free Ringer's solutionwere obtained from Wako Pure Chemical Industries, Ltd. The Ca²⁺-freeRinger's solution had a pH of 7.2.

The mouse head thus cooled was moved to a sherbet of a Ca²⁺-freeRinger's solution prepared in a deep petri dish. Subsequently, while themouse head was cooled, an olfactory epithelium tissue included in themouse head was isolated.

The isolated olfactory epithelium tissue was dispersed in a Ca²⁺-freeRinger's solution cooled on ice. This Ca²⁺-free Ringer's solution hadnot only the chemical reagents shown in the following Table 1 but alsothe chemical reagents shown in the following Table 2. In this way, anolfactory epithelium sample solution was obtained.

TABLE 2 Chemical reagent Concentration Available from Papain 1 unit/Sigma Aldrich milliliter Cystein 1 mM Wako Pure Chemical Industries,Ltd.

Then, the olfactory epithelium sample solution was stirred calmly undera room temperature with a rotator (available from AS ONE Corporation,commercial name: MTR-103). During this stir, decomposition reaction ofprotein was proceeded by the papain, which is a proteolytic enzyme.

Five minutes later, a Ringer's solution (1,600 microliters) containingthe chemical reagents shown in Table 3 was added to the olfactoryepithelium sample solution to stop the enzyme reaction of the papain.

TABLE 3 Chemical reagent Concentration Available from Leupeptin 500 μMSigma Aldrich Dnase I 200 unit/ Worthington milliliter Biochemical Co.BSA 0.1 mg/mL Sigma Aldrich NaCl 140 mM Wako Pure Chemical KCl 5 mMIndustries, Ltd. CaCl₂ 1 mM MgCl₂ 1 mM HEPES 10 mM Glucose 10 mMPyruvate sodium 1 mM

The Ca²⁺-free Ringer's solution had a pH of 7.2.

The olfactory epithelium sample solution was passed twice through a cellstrainer (available from BD Falcon company, 35 micrometers mesh). Inthis way, large tissue flakes was removed to obtain an olfactory cellsuspension.

The obtained olfactory cell suspension was subjected to a centrifugalseparation at an rpm (revolutions per minutes) of 1000 for five minutes.After the supernatant liquid was removed, three milliliters of theRinger's solution was newly added. In this way, an olfactory cell samplesolution was prepared.

(Step (a): Isolation of One Olfactory Cell)

The olfactory cell sample solution was supplied to a culture petri dish(available from BD falcon company) having a diameter of 60 millimeters.Subsequently, the olfactory cell sample solution was left at rest undera temperature of 37 degrees Celsius for 30 minutes. In this way, theolfactory cell was adsorbed on the culture petri dish.

One olfactory cell was collected from this culture petri dish with amicroscope (available from Olympus Corporation, a commercial name:IX-81). In more detail, a glass tube (available from Sutter InstrumentsCo., commercial name: B150-86-10) was steepled with a capillary tubepuller (available from Sutter Instruments Co., commercial name:P-97/IVF). The sharpened end of this glass tube had a diameter of 10micrometers.

Then, the Ringer's solution having the composition shown in Table 4 wasfilled by capillarity phenomenon into the sharpened end of the glasstube.

TABLE 4 Chemical reagent name Concentration Available from NaCl 140 mMWako Pure Chemical KCl 5 mM Industries, Ltd. CaCl₂ 1 mM MgCl₂ 1 mM HEPES10 mM Glucose 10 mM Pyruvate sodium 1 mM

The glass tube was installed with a microinjector (available fromEppendorf company, commercial name: CellTram vario) which was connectedto a manipulator (available from Narishige Co., Ltd., commercial name:MMN-1). After the one olfactory cell absorbed on the culture petri dishwas found with the microscope, the sharpened end of the glass tube wasset manually in the neighborhood of the one olfactory cell, using themanipulator. Subsequently, the one olfactory cell was collected into theglass tube with a microinjector by capillarity phenomenon.

(Step (b): Extraction of mRNA)

mRNA was extracted from the collected one olfactory cell. Thisextraction was carried out using a kit available from Takara Bio Inc. asa commercial name “CellAmp Whole Transcriptome Amplification Kit Ver2”.This kit was also used in the next step (c).

In more detail, the collected one olfactory cell was discharged into thePCR tube containing cell lysate (4.5 microliters) which was included inthe kit. The PCR tube was left under a temperature of 70 degrees Celsiusfor 90 seconds. In this way, the mRNA was obtained in the PCR tube.

(Step (c): Synthesis of cDNA)

According to the manual attached to the kit, cDNA was synthesized fromthe mRNA obtained in the step (b). In this way, an aqueous solution (25microliters) which contained the cDNA was obtained. Pure water of 225microliter was added to this aqueous solution.

(Step (d): Amplification of the cDNA)

PCR was performed using the cDNA obtained in the step (c), a forwardprimer represented by SEQ ID: 01, and a reverse primer represented bySEQ ID: 02.

Table 5 shows the composition of the solution used in this PCR.

TABLE 5 Volume (Unit: miicroliter) Concentration 2xGC buffer I 12.5 —(attached in the kit) dNTPs 4  0.4 mM (available from Takara bio inc.)Forward primer 1 1.2 μM Reverse primer 1 1.2 μM cDNA template 2.5 — Purewater 3.75 Takara LA Taq 0.25 Total 25

Table 6 shows the protocol of this PCR.

TABLE 6 Step Temperature Time 1 94 degrees Celsius One minute 2 94degrees Celsius 30 seconds 3 40 degrees Celsius 30 seconds 4 72 degreesCelsius Two minutes 5 After the steps 2-4 were repeated 40 times, thestep 6 was performed. 6 72 degrees Celsius 5 minutes 7  4 degreesCelsius 18 hours

After the PCR, a part of the PCR solution was subjected toelectrophoresis using an agarose gel. In this way, it was confirmed thata part of the gene coding for an olfactory receptor was amplified.

(Step (e))

The other part of the PCR solution was subjected to electrophoresisusing another agarose gel. Subsequently, the agarose gel was irradiatedwith ultra-violet rays to obtain plural bands derived from the PCRproducts. Then, a part of the agarose gel which contained each band wasisolated. The gene segment contained in the isolated agarose gel waspurified with a filter column (available from GL science, commercialname: MonoFas DNA purification kit). The forward primer represented bySEQ ID: 01 was added to the purified gene segment. Then, the genesequence of the gene segment was analyzed. As a result, it was foundthat the gene segment consisted of the gene sequence represented by SEQID: 10.

The BLAST search method was used to identify the gene sequencerepresented by SEQ ID: 10. For more detail of the BLAST search method,see the following home page: http://blast.ncbi.nlm.nih.gov/Blast.cgi.

As a result, the gene sequence represented by SEQ ID: 10 was identicalto the gene sequence olfr971.

(Step (f))

Therefore, the olfactory receptor included in the one olfactory cellobtained in a step (a) was identified as the olfactory receptor Olfr971.

Comparative Example 1

The experiment similar to the example 1 was conducted, except that apair of primers represented by SEQ ID: 03 and SEQ ID: 04 were usedinstead of the pair of the primers represented by SEQ ID: 01 and SEQ ID:02.

Comparative Example 2

The experiment similar to the example 1 was conducted, except that apair of primers represented by SEQ ID: 05 and SEQ ID: 07 were usedinstead of the pair of the primers represented by SEQ ID: 01 and SEQ ID:02.

Comparative Example 3

The experiment similar to the example 1 was conducted, except that apair of primers represented by SEQ ID: 06 and SEQ ID: 07 were usedinstead of the pair of the primers represented by SEQ ID: 01 and SEQ ID:02.

FIG. 2 shows an electrophoretic photograph in the neighborhood of 600base pairs in the comparative example 1, the comparative example 2, thecomparative example 3, and the example 1.

In FIG. 2, the column 1, the column 2, the column 3, and the column 4correspond to the comparative example 1, the comparative example 2, thecomparative example 3, and the example 1, respectively. The column 0 inFIG. 2 corresponds to a molecular weight marker.

As shown in FIG. 2, a band was observed in the neighborhood of 600 basepairs only in the example 1. This means that only the pair of theprimers represented by SEQ ID: 01 and SEQ ID: 02 achieve theamplification of the gene sequence olfr971.

Reference Example 1

The following reference example 1 was performed to determine which theolfactory receptor genes olfrs are amplified with the pair of theprimers represented by SEQ ID: 01 and SEQ ID: 02.

To start with the conclusion, the olfactory receptor genes olfrs whichcan be amplified by a PCR method using the pair of the primersrepresented by SEQ ID: 01 and SEQ ID: 02 are listed in the followingTable 7 as “Olfactory receptor gene group A”.

[Table 7]

(Olfactory Receptor Gene Group A)

olfr1, olfr8, olfr20, olfr22-ps1, olfr29-ps1, olfr30, olfr33, olfr44,olfr53, olfr76, olfr155, olfr156, olfr159, olfr209, olfr290, olfr291,olfr295, olfr314, olfr327-ps1, olfr329, olfr332, olfr361, olfr368,olfr373, olfr382, olfr437, olfr457, olfr472, olfr476, olfr480, olfr482,olfr484, olfr498, olfr502, olfr505-ps1, olfr508, olfr538, olfr544,olfr694, olfr695, olfr697, olfr706, olfr713, olfr715, olfr721-ps1,olfr735, olfr802, olfr877, olfr914, olfr921, olfr945, olfr951, olfr971,olfr988, olfr992, olfr994, olfr995, olfr1032, olfr1082, olfr1084,olfr1095, olfr1097, olfr1120, olfr1145, olfr1152, olfr1162, olfr1170,olfr1173, olfr1328, olfr1425, olfr1502, and olfr1504

(Total: seventy-two kinds)

The olfactory receptor genes olfrs listed in Table 7 corresponds to theolfactory receptors Olfrs of the mouse olfactory receptor group A.

(Preparation of the Mouse Genomic DNA)

One mouse C57/BL6J (female) was purchased from Japan SLC, Inc. The mousewas 3-5 weeks old. The mouse tail was cut. The cut mouse tail had alength of approximately 1 millimeter.

A mouse genomic DNA was prepared using a kit (available from Qiagencompany, commercial name: DNeasy Blood & Tissue Kit) from the separatedmouse tail.

A PCR was performed using this mouse genomic DNA, the forward primerrepresented by SEQ ID: 01, and the reverse primer represented by SEQ ID:02.

The DNA polymerase used in this PCR was obtained from Takara bio inc. asa commercial name “LA-Taq with GC Buffer”.

A PCR solution similar to the PCR solution shown in Table 5 wasprepared, except that the mouse genomic DNA having a concentration of 5nanograms/microliter was added instead of the template cDNA. Thissolution was used in the above-mentioned PCR.

The protocol used in the PCR was identical to the protocol shown inTable 6.

In this way, PCR products were obtained.

A part of the obtained PCR products was subjected to electrophoresisusing an agarose gel.

After the electrophoresis, the agarose gel was irradiated withultra-violet rays to find a band derived from the PCR products.Subsequently, a part of the agarose gel including the band was isolated.

The PCR product contained in the part of the isolated agarose gel waspurified with a filter column (available from GL science company,commercial name: MonoFas DNA purification kit).

The purified PCR products were composed of various types of genesegments. In order to determine the gene sequence of each gene segment,these gene segments were inserted into expression plasmids, and a TAcloning was performed with E. coli. The detail of the TA cloning isdescribed below.

A part of the purified PCR products, a plasmid (available from Takarabio inc., commercial name: pMD20-T), and ligase ((available from Takarabio inc., commercial name: DNA ligation kit ver1) were mixed in purewater. After mixing, the mixture was left under a temperature of 16degrees Celsius for 30 minutes. In this way, prepared were the plasmidswhere the purified PCR products, namely, the purified gene sequences,were inserted.

An aqueous E. Coli solution (available from Toyobo Co., Ltd., commercialname: DH5α, as E. Coli) was added to the aqueous solution containingthese plasmids to obtain a mixture. Subsequently, the mixture was leftunder a temperature of 4 degrees Celsius for 15 minutes. Furthermore,the mixture was left under a temperature of 42 degrees Celsius for 90seconds. Finally, the mixture was left under a temperature of 4 degreesCelsius for three minutes. In this way, the E. Coli was transformed withthe plasmids.

The E. Coli transformed with the plasmids was incubated under atemperature of 37 degrees Celsius for 15 hours on an LB plate (availablefrom InvivoGen company, commercial name “fas-am-x”).

The LB plate had a culture medium containing the composition shown infollowing Table 8.

TABLE 8 Chemical reagent Concentration Ampicillin 100micrograms/milliliter X-gal 100 micrograms/milliliter IPTG 100micrograms/milliliter

After the incubation, white colonies formed on the LB plate werecollected. The number of the collected colonies was approximately 110.Each colony was incubated under a temperature of 37 degrees Celsius for15 hours in the LB liquid medium containing ampicillin having aconcentration of 100 micrograms/microliter to obtain an E. Colisolution.

The plasmids where the gene segments were inserted were purified fromthe E. Coli solution with a plasmid purification kit (available fromQiagen company commercial name: DirectPrep 96 MiniPrep Kit).

A primer (available from Takara bio inc., commercial name: M13) wasadded to a part of the purified plasmid aqueous solution. Subsequently,the plasmid aqueous solution was commissioned to Greiner Bio-One Co.Ltd. (Japan) to analyze the gene sequence included in the PCR products.

The amplified olfactory receptor genes olfrs were identified by theBLAST search method on the basis of the gene sequence analyzed byGreiner Bio-One Co. Ltd. (Japan). As a result, it was found thatseventy-two kinds of the olfactory receptor genes olfrs shown in Table 7were amplified.

Reference Comparative Example 1

The experiment similar to the reference example 1 was conducted, exceptthat the pair of the primers represented by SEQ ID: 03 and SEQ ID: 04were used instead of the pair of the primers represented by SEQ ID: 01and SEQ ID: 02.

As a result, it was found that forty-seven kinds of the olfactoryreceptor genes olfrs were amplified. These forty-seven kinds of theolfactory receptor genes olfrs are shown in Table 9 as “Olfactoryreceptor gene group C1”.

[Table 9]

(Olfactory Receptor Gene Group C1)

olfr16, olfr30, olfr53, olfr90, olfr92, olfr132, olfr136, olfr155,olfr159, olfr160, olfr164, olfr287, olfr288, olfr317, olfr333, olfr374,olfr457, olfr483, olfr498, olfr517, olfr535, olfr713, olfr715, olfr764,olfr769, olfr796, olfr944, olfr977, olfr977-ps1, olfr982, olfr998,olfr1096, olfr1097, olfr1098, olfr1099, olfr1120, olfr1130, olfr1162,olfr1178, olfr1208, olfr1261, olfr1294, olfr1297, olfr1348, olfr1349,olfr1359, olfr1393, and olfr1507

Reference Comparative Example 2

The experiment similar to the reference example 1 was conducted, exceptthat the pair of the primers represented by SEQ ID: 05 and SEQ ID: 07were used instead of the pair of the primers represented by SEQ ID: 01and SEQ ID: 02.

As a result, it was found that twenty-six kinds of the olfactoryreceptor genes olfrs were amplified. These twenty-six kinds of theolfactory receptor genes olfrs are shown in Table 10 as “Olfactoryreceptor gene group C2”.

[Table 10]

(Olfactory Receptor Gene Group C2)

olfr2, olfr6, olfr32, olfr62, olfr119, olfr141, olfr155, olfr159,olfr220, olfr544, olfr545, olfr560, olfr566, olfr1010, olfr1095,olfr1126, olfr1129, olfr1130, olfr1150, olfr1257, olfr1274, olfr1342,olfr1347, olfr1348, olfr1402, and olfr1506

Reference Comparative Example 3

The experiment similar to the reference example 1 was conducted, exceptthat the pair of the primers represented by SEQ ID: 06 and SEQ ID: 07were used instead of the pair of the primers represented by SEQ ID: 01and SEQ ID: 02.

As a result, it was found that forty-three kinds of the olfactoryreceptor genes olfrs were amplified. These forty-three kinds of theolfactory receptor genes olfrs are shown in Table 11 as “Olfactoryreceptor gene group C3”.

[Table 11]

(Olfactory Receptor Gene Group C3)

olfr22, olfr23, olfr24, olfr49, olfr51, olfr62, olfr70, olfr133,olfr136, olfr138, olfr141, olfr228, olfr350, olfr373, olfr374, olfr382,olfr386, olfr389, olfr392, olfr393, olfr395, olfr397, olfr459, olfr483,olfr701, olfr711, olfr790, olfr796, olfr854, olfr860, olfr1126,olfr1129, olfr1155, olfr1328, olfr1338, olfr1342, olfr1355, olfr1424,olfr1425, olfr1491, olfr1496, olfr1507, and olfr1509

The gene sequences olfrs amplified only with the pair of the primersrepresented by SEQ ID: 01 and SEQ ID: 02 were extracted on the basis ofthe following formula. Hereinafter, the term “Olfactory receptor genegroup B” includes the olfactory genes olfrs amplified only with the pairof the primers represented by SEQ ID: 01 and SEQ ID: 02.

“Olfactory receptor gene group B”=“Olfactory receptor gene groupA”−(“Olfactory receptor gene group C1”+“Olfactory receptor gene groupC2”+“Olfactory receptor gene group C3”)

Needless to say, the olfactory receptor genes olfrs included in theolfactory receptor gene group B fails to be amplified with a pair ofprimers other than the pair of the primers represented by SEQ ID: 01 andSEQ ID: 02. The olfactory receptor gene group B corresponds to the mouseolfactory receptor group B.

The present invention provides a novel method for identifying anolfactory receptor included in one olfactory cell.

What is claimed is:
 1. A method for identifying an olfactory receptorincluded in one olfactory cell among a mouse olfactory receptor group A;the method comprising steps of: (a) obtaining the one olfactory cell;(b) extracting mRNA from the one olfactory cell obtained in the step(a); (c) obtaining cDNA with reverse transcriptase from the mRNAextracted in the step (b); (d) amplifying the cDNA obtained in the step(c) by a PCR method using a forward primer represented by SEQ ID: 01 anda reverse primer represented by SEQ ID: 02; wherein SEQ ID:01 is A(adenine) -T (thymine) -G (guanine) -K (guanine or thymine) -C(cytosine) -I (inosine) -T (thymine) -W (adenine or thymine) -I(inosine) -G (guanine) -A (adenine) -Y (thymine or cytosine) -M (adenineor cytosine) -G (guanine) -I (inosine) -T (thymine) -A (adenine) -Y(thymine or cytosine) -G (guanine) -T (thymine) -I (inosine) -G(guanine) -C (cytosine); and SEQ ID: 02 is T (thymine) -C (cytosine) -Y(thymine or cytosine) -T (thymine) -K (guanine or thymine) -R (guanineor adenine) -T (thymine) -T (thymine) -Y (thymine or cytosine) -C(cytosine) -T (thymine) -I (inosine) -A (adenine) -G (guanine) -R(guanine or adenine) -C (cytosine) -T (thymine) -R (guanine or adenine)-T (thymine) -A (adenine) -I (inosine) -A (adenine) -T (thymine) -I(inosine) -A (adenine) -G (guanine) -I (inosine) -G (guanine) -G(guanine) -R (guanine or adenine) -T (thymine) -T (thymine) (e)determining whether or not a gene sequence of the cDNA amplified in thestep (d) is identical to one gene sequence included in gene sequencescoding for olfactory receptors included in the mouse olfactory receptorgroup A; (f) determining that, if the gene sequence of the cDNA isidentical to the one gene sequence in the step (e), the olfactoryreceptor included in the one olfactory cell is the olfactory receptorcorresponding to the one gene sequence which is identical to the genesequence of the cDNA in the step (e); where the mouse olfactory receptorgroup A includes the following mouse olfactory receptors: (Mouseolfactory receptor group A) Olfr1, Olfr8, Olfr20, Olfr22-ps1,Olfr29-ps1, Olfr30, Olfr33, Olfr44, Olfr53, Olfr76, Olfr155, Olfr156,Olfr159, Olfr209, Olfr290, Olfr291, Olfr295, Olfr314, Olfr327-ps1,Olfr329, Olfr332, Olfr361, Olfr368, Olfr373, Olfr382, Olfr437, Olfr457,Olfr472, Olfr476, Olfr480, Olfr482, Olfr484, Olfr498, Olfr502,Olfr505-ps1, Olfr508, Olfr538, Olfr544, Olfr694, Olfr695, Olfr697,Olfr706, Olfr713, Olfr715, Olfr721-ps1, Olfr735, Olfr802, Olfr877,Olfr914, Olfr921, Olfr945, Olfr951, Olfr971, Olfr988, Olfr992, Olfr994,Olfr995, Olfr1032, Olfr1082, Olfr1084, Olfr1095, Olfr1097, Olfr1120,Olfr1145, Olfr1152, Olfr1162, Olfr1170, Olfr1173, Olfr1328, Olfr1425,Olfr1502 and Olfr1504.
 2. The method according to claim 1, furthercomprising: disrupting a cell membrane of the one olfactory cell betweenthe step (a) and the step (b).
 3. The method according to claim 1,wherein the one olfactory cell is extracted from a sample solutioncontaining olfactory cells in the step (a).
 4. The method according toclaim 1, wherein the mouse olfactory receptor group A is selected fromthe following mouse olfactory receptor group B: (Mouse olfactoryreceptor group B) Olfr 1, Olfr 8, Olfr 20, Olfr 22-ps1, Olfr 29-ps1,Olfr 33, Olfr 44, Olfr 76, Olfr156, Olfr209, Olfr290, Olfr291, Olfr295,Olfr314, Olfr327-ps1, Olfr329, Olfr332, Olfr361, Olfr368, Olfr437,Olfr472, Olfr476, Olfr480, Olfr482, Olfr484, Olfr502, Olfr505-ps1,Olfr508, Olfr538, Olfr694, Olfr695, Olfr697, Olfr706, Olfr721-ps1,Olfr735, Olfr802, Olfr877, Olfr914, Olfr921, Olfr945, Olfr951, Olfr971,Olfr988, Olfr992, Olfr994, Olfr995, Olfr1032, Olfr1082, Olfr1084,Olfr1145, Olfr1152, Olfr1170, Olfr1173, Olfr1502 and Olfr1504.